1. Field of the Invention
The subject of the present invention is a separation procedure for Lp(a) by means of electrophoresis, gels for the implementation of this procedure, as well as the application of this procedure to the determination of the atherogenic risk associated with the presence of Lp(a) and more particularly of the risks which an individual presents being affected by a disease such as atherosclerosis or any other disease associated with the latter.
2. Prior Art
The electrophoresis of human serum or plasma in agarose gel, followed by specific staining of the lipids, makes it possible to separate and visualize the different lipoprotein fractions, HDL, VLDL, LDL and chylomicrons, the levels of each of which are valuable indicators of the risks of atherosclerosis (FREDRICKSON et al., 1967, New Engl. J. Med., 276).
"Lp(a)" is a specific lipoprotein, identified by BERG in 1963 (Acta Pathol. Microbiol. Scand., 59, 369). It possesses a great structural similarity to the LDLs, but is characterized by an additional protein moiety, called apo(a), linked to the apo B100 by means of disulfide bridges.
Although the physiological role of Lp(a) has not been completely elucidated, many clinical studies have shown that a high level of plasma Lp(a) (higher than 0.3 g/l) constitutes an independent risk factor for atherogenesis (KOSTNER et al., 1981, Atherosclerosis, 38, 51; RHOADS et al. 1986, J. Ann. Med. Assoc., 256, 2540).
At present, classical electrophoresis of human serum or plasma (standard gel: 0.5% agarose, in Tris Veronal buffer; see FIG. 1A), usually allows the resolution of three lipoprotein fractions: the HDL (position .alpha.), the VLDL (position pre.beta.) and the LDL (position .beta.). The Ip(a) fraction, when it is present, migrates to the pre.beta.1 position, and, it usually cannot be adequately distinguished from the VLDL for its presence or absence to be concluded with certainty in the context of the determination of the atherogenic risk associated with the presence of Lp(a) (BRUCKERT et al., 1990, Clin. Chim. Acta, 188, 71).
Consequently, the different methods for the detection and analysis of plasma Lp(a) are, at present, all based on the use of specific antibodies. More particularly, these methods are the following: immunonephelometry (CAZZOLATO et al., 1983, Clin. Chim. Acta, 135, 203), electrosyneresis (MOLINARI et al., 1983, Clin. Chim. Acta, 128, 373), electroimmunodiffusion according to LAURELL (KOSTNER et al., 1981, Atherosclerosis, 33, 51), the ELISA immunoenzymatic procedures (ABE et al., 1988, Clin. Chim. Acta, 177, 31), as well as an immunolatex procedure (VU DAC et al., 1985, J. Lipid. Res., 26, 267).
However, these assays are usually expensive, and are not systematically prescribed by the clinician in the context of dyslipoproteinemia research. Prior to such an assay, it would thus be very interesting to be able to detect the presence of Lp(a) in pathological amounts during a routine lipoprotein analysis.
One of the aims of the present invention is precisely that of placing at the disposition of clinicians a procedure for the separation of Lp(a) from the other lipoproteins of the serum, the cost of which is markedly lower than that of the present procedures, and which consequently may be prescribed in the context of routine dislipoproteinemia research.
The object of the present invention is also to make possible the detection of Lp(a) in a reliable manner and, as a consequence, the reliable in vitro diagnosis of the risk of appearance in an individual of diseases associated with atherosclerosis.